PROJECT SUMMARY There is an urgent need to develop new antimicrobial strategies to combat the increasing occurrence of drug resistance in clinical pathogens. Current antibiotics act on a limited set of cellular processes, and the rate of new inhibitor discovery is rapidly declining. With the diminishing arsenal of useful antibiotics, other essential cellular targets must be explored. During infection, bacterial pathogens rapidly respond to changes in the host microenvironment by remodeling metabolism to promote growth. These ?metabolic adaptations? are crucial for pathogen survival and pathogenicity in vivo, and are thus a promising target space for antibiotic development. Positioned at a metabolic branch point to supply essential vitamins and isoprenoids, 1-deoxy-D- xylulose 5-phosphate synthase (DXPS) is likely a key player in bacterial metabolic adaptation during infection and thus a potential target. We have uncovered several unique features of DXPS structure and mechanism that suggest it plays a potentially larger role in bacterial metabolism, and can be selectively targeted. These insights have guided the development of tools to enable pharmacological probing of DXPS in bacteria. In the next phase, we will: 1) investigate the effects of targeting DXPS in the context of bacterial metabolic adaptation during infection, and validate DXPS as a target in vivo, 2) expose new opportunities for selective inhibition by studying the unique mechanism and conformational dynamics of DXPS, and 3) apply these insights to identify and understand other DXPS-dependent activities. These studies could lead to discovery of other targetable functions for antibiotic development.